Gene Silencing by RNA Interference: Technology and Application......Page 1
Foreword......Page 6
Preface......Page 7
Acknowledgments......Page 8
Editor......Page 9
Contributors......Page 10
Table of Contents......Page 14
1.1 THE IDENTIFICATION OF POST- TRANSCRIPTIONAL GENE SILENCING MECHANISMS......Page 17
1.2 RNA SILENCING, SIRNAS, AND RISC......Page 18
1.3 SPECIES- SPECIFIC ASPECTS OF RNA- TRIGGERED GENE SILENCING......Page 19
1.4 RNAI AND EPIGENETIC ENDOGENOUS RNA SILENCING......Page 21
1.5 APPLICATIONS OF RNAI BASED TECHNOLOGIES......Page 22
REFERENCES......Page 23
2.1 INTRODUCTION: SUCCESSFUL RNA INTERFERENCE DEPENDS ON DESIGN AND SYNTHESIS......Page 30
2.2 DESIGN......Page 31
2.2.1 RANDOM METHODS OF SIRNA DESIGN......Page 32
2.2.2 CONVENTIONAL METHODS OF SIRNA DESIGN......Page 34
2.2.3.1 Bioinformatically Controlled Pools of Rationally Designed siRNAs......Page 37
2.2.3.2 Rational siRNA Design: Potency and Longevity......Page 38
2.2.3.3 Rational siRNA Design: Specificity......Page 40
2.3 SIRNA PRODUCTION TECHNOLOGIES......Page 41
PROTOCOLS......Page 44
REFERENCES......Page 48
3.1 INTRODUCTION......Page 51
3.2.1 RNASE H MAPPING......Page 52
3.2.2 ANTISENSE OLIGONUCLEOTIDE ARRAYS......Page 53
3.3.1 DESIGN OF SIRNAS......Page 56
3.3.2 ASSESSMENT OF IGF1R GENE SILENCING IN HUMAN AND MURINE TUMOR CELLS......Page 57
3.4 CONCLUSIONS......Page 61
PROTOCOLS......Page 63
ACKNOWLEDGMENTS......Page 70
REFERENCES......Page 71
4.1 INTRODUCTION......Page 73
4.2 STRATEGY TO GENERATE SIRNAS BY USING IN VITRO TRANSCRIPTION......Page 74
4.3.1 CRITERIA......Page 76
4.4 INHIBITION OF HUMAN......Page 77
PROTOCOLS......Page 80
APPENDIX 4.1 TROUBLESHOOTING GUIDE......Page 84
REFERENCES......Page 85
5.1 INTRODUCTION......Page 87
5.2.2 PARTIALLY DOUBLE- STRANDED TEMPLATE COUPLED WITH KLENOW FILL-IN REACTION......Page 89
5.3 DESIGN OF DEOXYRIBOZYMES AND MODIFICATION OF THE T7 LEADER......Page 90
5.4 PRODUCTION OF SMALL INTERFERING RNAS......Page 91
5.4.2 IN VITRO TRANSCRIPTION......Page 92
5.4.3 DEOXYRIBOZYME DIGESTION AND PREPARATION OF TRANSFECTION-READY SIRNAS......Page 93
PROTOCOLS......Page 94
ACKNOWLEDGMENTS......Page 98
REFERENCES......Page 99
6.1 INTRODUCTION......Page 100
6.2 CLONING, EXPRESSION, AND PURIFICATION OF E. COLI RNASE III......Page 102
PROTOCOLS......Page 103
REFERENCES......Page 111
7.1 INTRODUCTION......Page 113
7.2.2.1 Choice of Promoter......Page 114
7.2.2.2 Selection and Modification of the Target Sites......Page 115
7.2.2.3 Cytoplasmic Expression of siRNA......Page 117
PROTOCOLS......Page 118
REFERENCES......Page 121
8.1 INTRODUCTION......Page 123
8.2 VIRAL VECTORS FOR......Page 124
8.3.1 DESIGN AND CONSTRUCTION OF LENTIVIRAL VECTORS FOR SHRNA DELIVERY AND EXPRESSION......Page 125
8.3.1.1 Effects on Viral Titer......Page 126
8.3.2.2 Generation of Transgenic Knockout Mouse Models......Page 127
8.3.2.3 Antiviral Applications and Gene Therapy......Page 128
8.3.2.4.2 Interferon Response in shRNA-Transduced Cells......Page 129
8.4 CONCLUDING REMARKS......Page 130
PROTOCOLS......Page 131
REFERENCES......Page 133
9.1 INTRODUCTION......Page 139
9.1.2 SIRNA DELIVERY SYSTEMS......Page 140
9.2.1 THE OPTIMIZATION STRATEGY......Page 145
PROTOCOLS......Page 148
REFERENCES......Page 155
10.1 INTRODUCTION TO THE USE OF RNA INTERFERENCE IN DROSOPHILA-CULTURED CELLS......Page 158
10.2 CHOOSING A CELL LINE AND CULTURING DROSOPHILA CELLS......Page 159
10.3.1 PREPARATION OF THE TEMPLATE FOR TRANSCRIPTION......Page 160
10.3.2 IN VITRO TRANSCRIPTION AND DETERMINATION
OF DSRNA CONCENTRATION......Page 163
10.4.2 TRANSFECTION OF OTHER CELL LINES......Page 164
10.4.3 ANALYSIS OF THE TRANSFECTION EFFICIENCY AND CELLS AFTER TRANSFECTION......Page 165
10.5 LARGE- SCALE RNAI......Page 166
10.5.2.2 PCR Amplification of Gene- Specific Sequence......Page 167
10.5.2.3 dsRNA Preparation......Page 168
10.5.4. ANALYSIS OF PHENOTYPE IN LARGE-SCALE SCREENS......Page 169
PROTOCOLS......Page 170
REFERENCES......Page 175
11.1 INTRODUCTION......Page 178
11.2.2 BACTERIA EXPRESSING DSRNA......Page 180
11.2.3 TRANSGENE-DERIVED DSRNA......Page 181
11.3.1 INJECTION OF DSRNA......Page 182
11.3.3 FEEDING OF BACTERIA EXPRESSING DSRNA......Page 183
11.3.4 TRANSGENIC WORMS EXPRESSING DSRNA......Page 184
11.3.5.3 Zygotic RNAi......Page 185
11.3.5.5 Hypersensitive Strains......Page 186
11.4 APPLICATION AND READ- OUT......Page 187
11.5 LIMITATIONS OF RNAI......Page 188
PROTOCOLS......Page 189
REFERENCES......Page 191
12.1 INTRODUCTION......Page 194
12.3.1 PREPARATION OF DSRNA......Page 196
12.3.2.2 Preparation of Microinjection Needles......Page 197
12.3.2.3 Gonad Microinjection......Page 199
12.4 ANALYSIS OF PHENOTYPES......Page 202
12.5 USEFUL CONTROL EXPERIMENTS......Page 203
PROTOCOLS......Page 204
REFERENCES......Page 210
13.1 INTRODUCTION......Page 212
13.2 DESIGNING A DSRNA TRIGGER......Page 213
13.2.1 THE SIRNA OPTION......Page 214
13.3.1 NOTE ON NEEDLE PREPARATION......Page 215
13.3.2 EMBRYO PREPARATION......Page 216
13.5 EXPECTED RESULTS......Page 217
13.6 PROBLEMS AND PITFALLS......Page 218
PROTOCOLS......Page 219
REFERENCES......Page 224
14.1 INTRODUCTION......Page 226
14.2 RNAI OF GENES ENCODING ENDOGENOUS XENOPUS OOCYTE ION CHANNEL b SUBUNITS......Page 227
14.3 CONCLUSION......Page 229
PROTOCOLS......Page 230
ACKNOWLEDGMENTS......Page 239
REFERENCES......Page 240
15.1 INTRODUCTION......Page 241
PROTOCOLS......Page 242
REFERENCES......Page 248
16.1 INTRODUCTION......Page 251
16.2 THE PLASMID PZJMa - A VECTOR FOR RNAI IN T. BRUCEI......Page 252
16.3 FORWARD GENETICS USING AN RNAI- BASED GENOMIC LIBRARY......Page 253
PROTOCOLS......Page 256
OTHER SOLUTIONS......Page 265
REFERENCES......Page 266
17.1 INTRODUCTION......Page 268
17.2.1 THE TARGET GENE AND PARASITE STAGE......Page 269
17.2.3 DELIVERY SYSTEM......Page 271
17.3 DELIVERY TECHNIQUES......Page 272
PROTOCOLS......Page 274
REFERENCES......Page 277
18.1 INTRODUCTION......Page 279
18.2 RNAI IN PARASITES AND LOWER ORGANISMS......Page 280
18.3 RNAI IN PLASMODIUM......Page 281
18.6 CONCLUDING REMARKS......Page 284
PROTOCOLS......Page 285
REFERENCES......Page 293
19.1 INTRODUCTION......Page 296
PROTOCOLS......Page 297
REFERENCES......Page 304
20.1 INTRODUCTION......Page 305
20.2 PROTOCOLS FOR DELIVERING SIRNAS AS DRUGS......Page 306
PROTOCOLS......Page 307
REFERENCES......Page 325
21.1 INTRODUCTION......Page 328
21.2 RNA SILENCING AS A SPECIFIC PLANT IMMUNE SYSTEM......Page 329
21.3 VIGS......Page 330
21.4.1 VIRAL VECTORS......Page 334
21.4.3 VIGS ASSESSMENT......Page 336
PROTOCOLS......Page 337
REFERENCES......Page 345
22.1 INTRODUCTION......Page 348
22.3 GENES INVOLVED IN RNA SILENCING......Page 349
22.4 MAINTENANCE OF THE SILENT STATE: PRODUCTION OF SECONDARY SIRNAS AND TRANSITIVITY......Page 351
22.6 RNA- DEPENDENT DNA METHYLATION......Page 352
22.7 EXPERIMENTAL INDUCTION OF RNA SILENCING......Page 353
PROTOCOL......Page 354
REFERENCES......Page 356
23.1 INTRODUCTION......Page 361
23.2 AGRO- INFILTRATION- MEDIATED IDENTIFICATION OF RNAI SUPPRESSOR PROTEINS......Page 362
23.3 OTHER APPLICATIONS OF AGRO- INFILTRATION IN THE ANALYSIS OF RNAI IN PLANTS......Page 363
PROTOCOLS......Page 364
REFERENCES......Page 367